This R03 grant proposal is designed to provide the "proof of principle" of our hypothesis-driven project and is therefore the solid ground for an NIH R01 application. Systemic lupus erythematosus (SLE) is characterized by a unique spectrum of pathogenic autoantibodies, mainly directed at ubiquitous nuclear targets. The most frequent autoantigens are nucleosomes, the fundamental structure of chromatin, and their components: double stranded DNA (dsDNA) and histones. To understand the etiology of SLE, it is important to know the source of nucleosomal DNA and how it incites the autoimmune response. Because impaired clearance of apoptotic cells or their debris can lead to SLE-like disease in certain mouse models, because SLE autoantigens concentrate on apoptotic blebs and bodies, and because generation of nucleosomal DNA is one of the most distinct characteristics of the apoptotic process, apoptotic cells have been proposed as a critical reservoir of autoantigens. We intend to use a genetically engineered mouse in which the ability to fragment DNA is disrupted, and therefore nucleosomal DNA of apoptotic origin is absent, as a tool to investigate in vivo if apoptotic cells are the main source of nucleosomes in SLE. We propose a time line of three years in which 2 AIMs will be attained. In AIM I, we will investigate chromatin redistribution, concentration into bodies and blebs, and release during apoptosis in vivo, in the absence of caspase activated DNase (CAD), the most important known DNase involved in the generation of apoptotic nucleosomal DNA. In AIM II we propose to generate several murine lupus models, which "lack" nucleosomal DNA of apoptotic origin because they are CAD deficient, and therefore cannot fragment DNA during apoptosis, to directly demonstrate that apoptotic nucleosomes are the driving autoantigen in SLE, and that they are necessary to develop and/or sustain the autoimmune response in lupus. The sets of experiments in AIM I and II will provide the initial important steps toward an R01 proposal application, which long term goals are to genetically manipulate chromatin fragmentation in order to manipulate lupus disease, and ultimately to use novel technologies to silence in vivo CAD, or other genes and so inhibit the generation of apoptotic nucleosomes as a potential treatment of this life threatening disease.